Identification of P2X receptors in cultured mouse and rat parasympathetic otic ganglion neurones including P2X knockout studies

We have used patch-clamp recording from cultured neurones, immunohistochemistry and gene deletion techniques to characterize the P2X receptors present in mouse otic ganglion neurones, and demonstrated the presence of similar receptors in rat neurones. All neurones from wild-type (WT) mice responded to ATP (EC(50) 109 microM), but only 38% also responded to alpha beta-meATP (EC(50) 39 microM). The response to alpha beta-meATP was blocked by TNP-ATP with an IC(50) of 38.6 nM. Lowering extracellular pH and co-application of Zn(2+) potentiated responses to ATP and alpha beta-meATP. In P2X(3)(-/-) mouse otic ganglion, all neurones tested responded to 100 microM ATP with a sustained current, but none responded to alpha beta-meATP. In P2X(2)(-/-) mice, no sustained currents were observed, but 36% of neurones responded to both ATP and alpha beta-meATP with transient currents. In P2X(2)/P2X(3)(Dbl-/-) mice, no responses to ATP or alpha beta-meATP were detected, suggesting that other P2X subunits were not involved. In rat otic ganglia, 96% of neurones responded to both ATP and alpha beta-meATP with sustained currents, suggesting a greater proportion of neurones expressing P2X(2/3) receptors. The maximum response to alpha beta-meATP was 40-60% of that evoked by ATP in the same cell. Immunohistochemistry revealed staining for P2X(2) and P2X(3) subunits in WT mouse otic ganglion neurones, which was absent in knockout animals. In conclusion, we have shown for the first time that at least two distinct P2X receptors are present in mouse and rat otic neurones, probably homomeric P2X(2) and heteromeric P2X(2/3) receptors.